IKK-α proteins, nucleic acids and methods

ABSTRACT

The invention provides methods and compositions relating to an IκB kinase, IKK-α, and related nucleic acids. The polypeptides may be produced recombinantly from transformed host cells from the disclosed IKK-α encoding nucleic acids or purified from human cells. The invention provides isolated IKK-α hybridization probes and primers capable of specifically hybridizing with the disclosed IKK-α genes, IKK-α-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

This is a divisional application of U.S. Ser. No. 08/890,854, filed Jul.10, 1997, which is a continuing application under 35USC120 of U.S. Ser.No. 08/887,115 filed Jul. 1, 1997, abandoned.

INTRODUCTION

1. Field of the Invention

The field of this invention is proteins involved in transcription factoractivation.

2. Background

Cytokines trigger changes in gene expression by modifying the activityof otherwise latent transcription factors (Hill and Treisman, 1995).Nuclear factor κB (NF-κB) is a prominent example of how such an externalstimulus is converted into an active transcription factor (Verma et al.,1995). The NF-κB system is composed of homo- and heterodimers of membersof the Rel family of related transcription factors that control theexpression of numerous immune and inflammatory response genes as well asimportant viral genes (Lenardo and Baltimore, 1989; Baeuerle and Henkel,1994). The activity of NF-κB transcription factors is regulated by theirsubcellular localization (Verma et al., 1995). In most cell types, NF-κBis present as a heterodimer comprising of a 50 kDa and a 65 kDa subunit.This heterodimer is sequestered in the cytoplasm in association withIκBα a member of the IκB family of inhibitory proteins (Finco andBaldwin, 1995; Thanos and Maniatis, 1995; Verma et al., 1995). IκBαmasks the nuclear localization signal of NF-κB and thereby preventsNF-κB nuclear translocation. Conversion of NF-κB into an activetranscription factor that translocates into the nucleus and binds tocognate DNA sequences requires the phosphorylation and subsequentubiquitin-dependent degradation of IκBα in the 26s proteasome.Signal-induced phosphorylation of IκBα occurs at serines 32 and 36.Mutation of one or both of these serines renders IκBα resistant toubiquitination and proteolytic degradation (Chen et al., 1995);

The pleiotropic cytokines tumor necrosis factor (TNF) and interleukin-1(IL-1) are among the physiological inducers of IκB phosphorylation andsubsequent NF-κB activation (Osborn et al., 1989; Beg et al., 1993).Although TNF and IL-1 initiate signaling cascades leading to NF-κBactivation via distinct families of cell-surface receptors (Smith etal., 1994; Dinarello, 1996), both pathways utilize members of the TNFreceptor-associated factor (TRAF) family of adaptor proteins as signaltransducers (Rothe et al., 1995; Hsu et al., 1996; Cao et al., 1996b).TRAF proteins were originally found to associate directly with thecytoplasmic domains of several members of the TNF receptor familyincluding the 75 kDa TNF receptor (TNFR2), CD40, CD30, and thelymphotoxin-β receptor (Rothe et al., 1994; Hu et al., 1994; Cheng etal., 1995; Mosialos et al., 1995; Song and Donner, 1995; Sato et al.,1995; Lee et al., 1996; Gedrich et al., 1996; Ansieau et al., 1996). Inaddition, TRAF proteins are recruited indirectly to the 55 kDa TNFreceptor (TNFR1) by the adaptor protein TRADD (Hsu et al., 1996).Activation of NF-κB by TNF requires TRAF2 (Rothe et al., 1995; Hsu etal., 1996). TRAF5 has also been implicated in NF-κB activation bymembers of the TNF receptor family (Nakano et al., 1996). In contrast,TRAF6 participates in NF-κB activation by IL-1 (Cao et al., 1996b). UponIL-1 treatment, TRAF6 associates with IRAK, a serine-threonine kinasethat binds to the IL-1 receptor complex (Cao et al., 1996a).

The NF-κB-inducing kinase (NIK) is a member of the MAP kinase kinasekinase (MAP3K) family that was identified as a TRAF2-interacting protein(Malinin et al., 1997). NIK activates NF-κB when overexpressed, andkinase-inactive mutants of NIK comprising its TRAF2-interactingC-terminal domain (NIK₍₆₂₄₋₉₄₇₎) or lacking two crucial lysine residuesin its kinase domain (NIK_((KK429-430AA))) behave as dominant-negativeinhibitors that suppress TNF-, IL-1-, and TRAF2-induced NF-κB activation(Malinin et al., 1997). Recently, NIK was found to associate withadditional members of the TRAF family, including TRAF5 and TRAF6.Catalytically inactive mutants of NIK also inhibited TRAF5- andTRAF6-induced NF-κB activation, thus providing a unifying concept forNIK as a common mediator in the NF-κB signaling cascades triggered byTNF and IL-1 downstream of TRAFs.

Here, we disclose a novel human kinase IκB Kinase, IKK-α, as aNIK-interacting protein. IKK-α has sequence similarity to the conceptualtranslate of a previously identified open reading frame (SEQ ID NO:5)postulated to encode a serine-threonine kinase of unknown function(‘Conserved Helix-loop-helix Ubiquitous Kinase’ or CHUK, Connelly andMarcu, 1995; Mock et al., 1995). Catalytically inactive mutants of IKK-αare shown to suppress NF-κB activation induced by TNF and IL-1stimulation as well as by TRAF and NIK overexpression; transientlyexpressed IKK-α is shown to associate with the endogenous IκBα complex;and IKK-α is shown to phosphorylate IκBα on serines 32 and 36.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to isolatedIKK-α polypeptides, related nucleic acids, polypeptide domains thereofhaving IKK-α-specific structure and activity and modulators of IKK-αfunction, particularly IκB kinase activity. IKK-α polypeptides canregulate NFκB activation and hence provide important regulators of cellfunction. The polypeptides may be produced recombinantly fromtransformed host cells from the subject IKK-α polypeptide encodingnucleic acids or purified from mammalian cells. The invention providesisolated IKK-α hybridization probes and primers capable of specificallyhybridizing with the disclosed IKK-α gene, IKK-α-specific binding agentssuch as specific antibodies, and methods of making and using the subjectcompositions in diagnosis (e.g. genetic hybridization screens for IKK-αtranscripts), therapy (e.g. IKK-α kinase inhibitors to inhibit TNFsignal transduction) and in the biopharmaceutical industry (e.g. asimmunogens, reagents for isolating other transcriptional regulators,reagents for screening chemical libraries for lead pharmacologicalagents, etc.).

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequence of a natural cDNA encoding a human IKK-αpolypeptide is shown as SEQ ID NO:3, and the full conceptual translateis shown as SEQ ID NO:4. The IKK-α polypeptides of the invention includeincomplete translates of SEQ ID NO:3 which translates and deletionmutants of SEQ ID NO:4 have human IKK-α-specific amino acid sequence,binding specificity or function and comprise at least one of Cys30,Leu604, Thr679, Ser680, Pro684, Thr686 and Ser678. Preferredtranslates/deletion mutants comprise at least a 6 residue Cys30, Leu604,Thr679, Ser680, Pro684, Thr686 or Ser687-containing domain of SEQ IDNO:4, preferably including at least 8, more preferably at least 12, mostpreferably at least 20 contiguous residues which immediately flank saidresidue, with said residue preferably residing within said contigousresidues, see, e.g. Table I; which mutants provide hIKK-α specificepitopes and immunogens.

TABLE I Exemplary Deletion Mutants Δ1 SEQ ID NO: 4, residues 22-31 Δ2SEQ ID NO: 4, residues 1-30 Δ3 SEQ ID NO: 4, residues 599-608 Δ4 SEQ IDNO: 4, residues 601-681 Δ5 SEQ ID NO: 4, residues 604-679 Δ6 SEQ ID NO:4, residues 670-687 Δ7 SEQ ID NO: 4, residues 679-687 Δ8 SEQ ID NO: 4,residues 680-690 Δ9 SEQ ID NO: 4, residues 684-695 Δ10 SEQ ID NO: 4,residues 686-699

The subject domains provide IKK-α domain specific activity or function,such as IKK-α-specific kinase or kinase inhibitory activity, NIK-bindingor binding inhibitory activity, IκB-binding or binding inhibitoryactivity, NFκB activating or inhibitory activity or antibody binding.Preferred domains phosphorylate at least one and preferably both theserine 32 and 36 of IκB (Verma, I. M., et al. (1995)). As used herein,Ser32 and Ser36 of IκB refers collectively to the two serine residueswhich are part of the consensus sequence DSGL/IXSM/L (e.g. ser 32 and 36in IκBα, ser 19 and 23 in IκBε, and ser 157 and 161, or 18 and 22,depending on the usage of methionines, in IκBε, respectively.

IKK-α-specific activity or function may be determined by convenient invitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cellculture assays, in animals (e.g. gene therapy, transgenics, etc.), etc.Binding assays encompass any assay where the molecular interaction of anIKK-α polypeptide with a binding target is evaluated. The binding targetmay be a natural intracellular binding target such as an IKK-αsubstrate, a IKK-α regulating protein or other regulator that directlymodulates IKK-α activity or its localization; or non-natural bindingtarget such a specific immune protein such as an antibody, or an IKK-αspecific agent such as those identified in screening assays such asdescribed below. IKK-α-binding specificity may assayed by kinaseactivity or binding equilibrium constants(usually at least about 10⁷M⁻¹, preferably at least about 10⁸ M⁻¹, more preferably at least about10⁹ M⁻¹), by the ability of the subject polypeptide to function asnegative mutants in IKK-α-expressing cells, to elicit IKK-α specificantibody in a heterologous host (e.g a rodent or rabbit), etc. In anyevent, the IKK-α binding specificity of the subject IKK-α polypeptidesnecessarily distinguishes the murine and human CHUK sequences ofConnelly and Marcu (1995) as well as IKK-β (SEQ ID NO:4).

The claimed IKK-α polypeptides are isolated or pure: an “isolated”polypeptide is unaccompanied by at least some of the material with whichit is associated in its natural state, preferably constituting at leastabout 0.5%, and more preferably at least about 5% by weight of the totalpolypeptide in a given sample and a pure polypeptide constitutes atleast about 90%, and preferably at least about 99% by weight of thetotal polypeptide in a given sample. The IKK-α polypeptides andpolypeptide domains may be synthesized, produced by recombinanttechnology, or purified from mammalian, preferably human cells. A widevariety of molecular and biochemical methods are available forbiochemical synthesis, molecular expression and purification of thesubject compositions, see e.g. Molecular Cloning, A Laboratory Manual(Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols inMolecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, NY) or that are otherwise known in the art.

The invention provides binding agents specific to IKK polypeptides,preferably the claimed IKK-α polypeptides, including substrates,agonists, antagonists, natural intracellular binding targets, etc.,methods of identifying and making such agents, and their use indiagnosis, therapy and pharmaceutical development. For example, specificbinding agents are useful in a variety of diagnostic and therapeuticapplications, especially where disease or disease prognosis isassociated with improper utilization of a pathway involving the subjectproteins, e.g. NF-κB activation. Novel IKK-specific binding agentsinclude IKK-specific receptors, such as somatically recombinedpolypeptide receptors like specific antibodies or T-cell antigenreceptors (see, e.g Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory) and other natural intracellularbinding agents identified with assays such as one-, two- andthree-hybrid screens, non-natural intracellular binding agentsidentified in screens of chemical libraries such as described below,etc. Agents of particular interest modulate IKK function, e.g.IKK-dependent transcriptional activation. For example, a wide variety ofinhibitors of IKK IκB kinase activity may be used to regulate signaltransduction involving IκB. Exemplary IKK IκB kinase inhibitors includeknown classes of serine/threonine kinase (e.g. PKC) inhibitors such ascompetitive inhibitors of ATP and substrate binding, antibiotics,IKK-derived peptide inhibitors, etc., see Tables II and III. IKKspecificity and activity are readily quantified in high throughputkinase assays using panels of protein kinases (see cited references andExamples).

Preferred inhibitors include natural compounds such as staurosporine(Omura S, et al. J Antibiot (Tokyo) 1995 July;48(7): 535-48), producedby a marine organism, and synthetic compounds such as PD 153035, whichalso potently inhibits the EGF receptor protein kinase (Fry D W et al.Science Aug. 19, 1994;265(5175): 1093-5). Members of the tyrphostinfamily of synthetic protein kinase inhibitors are also useful; theseinclude compounds which are pure ATP competitors, compounds which arepure substrate competitors, and compounds which are mixed competitors:compete with both ATP and substrate (Levitzki A and Gazit A, ScienceMar. 24, 1995;267(5205): 1782-8). Additional IKK inhibitors includepeptide-based substrate competitors endogenously made by the mammaliancell, e.g. PKI (protein kinase inhibitor, Seasholtz AF et al., Proc NatlAcad Sci USA Feb. 28, 1995;92(5): 1734-8), or proteins inhibiting cdckinases (Correa-Bordes J and Nurse P, Cell Dec. 15, 1995;83(6): 1001-9).Additional small peptide based substrate competitive kinase inhibitorsand allosteric inhibitors (inhibitory mechanisms independent of ATP orsubstrate competition) are readily generated by established methods(Hvalby O, et al. Proc Natl Acad Sci USA May 24, 1994;91(11): 4761-5;Barja P, et al., Cell Immunol 1994 January; 153(1):28-38; Villar-PalasiC, Biochim Biophys Acta Dec. 30, 1994;1224(3): 384-8; Liu WZ, et al.,Biochemistry Aug. 23, 1994;33(33): 10120-6).

TABLE II Selected Small Molecule IKK Kinase Inhibitors InhibitorsCitations HA-100¹ 1. Hagiwara, M., et al. Mol. Pharmacol. 32:7 (1987)Chelerythrine² 2. Herbert, J. M., et al. Biochem Biophys Res Com 172:993(1990) Staurosporine^(3,4,5) 3. Schachtele, C., et al. Biochem BiophysRes Com 151:542 (1988) Calphostin C^(6,7,8,9) 4. Tamaoki, T., et al.Biochem Biophys Res Com 135:397 (1986) K-252b¹⁰ 5. Tischler, A. S., etal. J. Neurochemistry 55:1159 (1990) PKC 19-36¹¹ 6. Bruns, R. F., et al.Biochem Biophys Res Com 176:288 (1991) Iso-H7¹² 7. Kobayashi, E., et al.Biochem Biophys Res Com 159:548 (1989) PKC 19-31 8. Tamaoki, T., et alAdv2nd Mass Phosphoprotein Res 24:497 (1990) H-7^(13,3,14) 9. Tamaoki,T., et al. Biotechnology 8:732 (1990) H-89¹⁵ 10. Yasuzawa, T. J.Antibiotics 39:1972 (1986) KT5720¹⁶ 11. House, C., et al. Science238:1726 (1987) cAMP-depPKinhib¹⁷ 12. Quick, J., et al. Biochem.Biophys. Res. Com. 167:657 (1992) A-3¹⁸ 13. Bouli, N. M. and Davis, M.Brain Res. 525:198 (1990) HA1004^(19,20) 14. Takahashi, I., et al. J.Pharmacol. Exp. Ther. 255:1218 (1990) K-252a^(16,5) 15. Chijiwa, T., etal. J. Biol. Chem. 265:5267 (1990) KT5823¹⁶ 16. Kase, H., et al.Biochem. Biophys. Res. Com. 142:436 (1987) ML-9²¹ 17. Cheng, H. C., etal. J. Biol. Chem. 261:989 (1986) KT5926²² 18. Inagaki, M., et al. Mol.Pharmacol. 29:577 (1986) 19. Asano, T. and Hidaka, H. J Pharmaco. ExpTher 231:141 (1984) 20. Hidaka, H., et al. Biochemistry 23:5036 (1984)21. Nagatsu, T., et al. Biochem Biophys Res Com 143:1045 (1987) 22.Nakanishi, S., et al. Mol. Pharmacol. 37:482 (1990)

TABLE II Selected Small Molecule IKK Kinase Inhibitors InhibitorsCitations HA-100¹ 1. Hagiwara, M., et al. Mol. Pharmacol. 32:7 (1987)Chelerythrine² 2. Herbert, J. M., et al. Biochem Biophys Res Com 172:993(1990) Staurosporine^(3,4,5) 3. Schachtele, C., et al. Biochem BiophysRes Com 151:542 (1988) Calphostin C^(6,7,8,9) 4. Tamaoki, T., et al.Biochem Biophys Res Com 135:397 (1986) K-252b¹⁰ 5. Tischler, A. S., etal. J. Neurochemistry 55:1159 (1990) PKC 19-36¹¹ 6. Bruns, R. F., et al.Biochem Biophys Res Com 176:288 (1991) Iso-H7¹² 7. Kobayashi, E., et al.Biochem Biophys Res Com 159:548 (1989) PKC 19-31 8. Tamaoki, T., et alAdv2nd Mass Phosphoprotein Res 24:497 (1990) H-7^(13,3,14) 9. Tamaoki,T., et al. Biotechnology 8:732 (1990) H-89¹⁵ 10. Yasuzawa, T. J.Antibiotics 39:1972 (1986) KT5720¹⁶ 11. House, C., et al. Science238:1726 (1987) cAMP-depPKinhib¹⁷ 12. Quick, J., et al. Biochem.Biophys. Res. Com. 167:657 (1992) A-3¹⁸ 13. Bouli, N. M. and Davis, M.Brain Res. 525:198 (1990) HA1004^(19,20) 14. Takahashi, I., et al. J.Pharmacol. Exp. Ther. 255:1218 (1990) K-252a^(16,5) 15. Chijiwa, T., etal. J. Biol. Chem. 265:5267 (1990) KT5823¹⁶ 16. Kase, H., et al.Biochem. Biophys. Res. Com. 142:436 (1987) ML-9²¹ 17. Cheng, H. C., etal. J. Biol. Chem. 261:989 (1986) KT5926²² 18. Inagaki, M., et al. Mol.Pharmacol. 29:577 (1986) 19. Asano, T. and Hidaka, H. J Pharmaco. ExpTher 231:141 (1984) 20. Hidaka, H., et al. Biochemistry 23:5036 (1984)21. Nagatsu, T., et al. Biochem Biophys Res Com 143:1045 (1987) 22.Nakanishi, S., et al. Mol. Pharmacol. 37:482 (1990)

Accordingly, the invention provides methods for modulating signaltransduction involving IκB in a cell comprising the step of modulatingIKK kinase activity, e.g. by contacting the cell with a serine/threoninekinase inhibitor. The cell may reside in culture or in situ, i.e. withinthe natural host. Preferred inhibitors are orally active in mammalianhosts. For diagnostic uses, the inhibitors or other IKK binding agentsare frequently labeled, such as with fluorescent, radioactive,chemiluminescent, or other easily detectable molecules, eitherconjugated directly to the binding agent or conjugated to a probespecific for the binding agent.

The amino acid sequences of the disclosed IKK-α polypeptides are used toback-translate IKK-α polypeptide-encoding nucleic acids optimized forselected expression systems (Holler et al. (1993) Gene 136, 323-328;Martin et al. (1995) Gene 154, 150-166) or used to generate degenerateoligonucleotide primers and probes for use in the isolation of naturalIKK-α-encoding nucleic acid sequences (“GCG” software, Genetics ComputerGroup, Inc, Madison Wis.). IKK-α-encoding nucleic acids used inIKK-α-expression vectors and incorporated into recombinant host cells,e.g. for expression and screening, transgenic animals, e.g. forfunctional studies such as the efficacy of candidate drugs for diseaseassociated with IKK-α-modulated cell function, etc.

The invention also provides nucleic acid hybridization probes andreplication/amplification primers having a IKK-α cDNA specific sequencecomprising at least 12, preferably at least 24, more preferably at least36 and most preferably at least contiguous 96 bases of a strand of SEQID NO:3 and including at least one of bases 1-92, 1811, 1812, 1992,1995, 2034, 2035, 2039, 2040, 2050, 2055 and 2060, and sufficient tospecifically hybridize with a second nucleic acid comprising thecomplementary strand of SEQ ID NO:3 in the presence of a third nucleicacid comprising (SEQ ID NO:5). Demonstrating specific hybridizationgenerally requires stringent conditions, for example, hybridizing in abuffer comprising 30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄,pH7.7, 0.001 M EDTA) buffer at a temperature of 42° C. and remainingbound when subject to washing at 42° C. with 0.2×SSPE; preferablyhybridizing in a buffer comprising 50% formamide in 5×SSPE buffer at atemperature of 42° C. and remaining bound when subject to washing at 42°C. with 0.2×SSPE buffer at 42° C. IKK-α nucleic acids can also bedistinguished using alignment algorithms, such as BLASTX (Altschul etal. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).

The subject nucleic acids are of synthetic/non-natural sequences and/orare isolated, i.e. unaccompanied by at least some of the material withwhich it is associated in its natural state, preferably constituting atleast about 0.5%, preferably at least about 5% by weight of totalnucleic acid present in a given fraction, and usually recombinant,meaning they comprise a non-natural sequence or a natural sequencejoined to nucleotide(s) other than that which it is joined to on anatural chromosome. Recombinant nucleic acids comprising the nucleotidesequence of SEQ ID NO:3, or requisite fragments thereof, contain suchsequence or fragment at a terminus, immediately flanked by (i.e.contiguous with) a sequence other than that which it is joined to on anatural chromosome, or flanked by a native flanking region fewer than 10kb, preferably fewer than 2 kb, which is at a terminus or is immediatelyflanked by a sequence other than that which it is joined to on a naturalchromosome. While the nucleic acids are usually RNA or DNA, it is oftenadvantageous to use nucleic acids comprising other bases or nucleotideanalogs to provide modified stability, etc.

The subject nucleic acids find a wide variety of applications includinguse as translatable transcripts, hybridization probes, PCR primers,diagnostic nucleic acids, etc.; use in detecting the presence of IKK-αgenes and gene transcripts and in detecting or amplifying nucleic acidsencoding additional IKK-α homologs and structural analogs. In diagnosis,IKK-α hybridization probes find use in identifying wild-type and mutantIKK-α alleles in clinical and laboratory samples. Mutant alleles areused to generate allele-specific oligonucleotide (ASO) probes forhigh-throughput clinical diagnoses. In therapy, therapeutic IKK-αnucleic acids are used to modulate cellular expression or intracellularconcentration or availability of active IKK-α.

The invention provides efficient methods of identifying agents,compounds or lead compounds for agents active at the level of a IKKmodulatable cellular function. Generally, these screening methodsinvolve assaying for compounds which modulate IKK interaction with anatural IKK binding target, in particular, IKK phosphorylation ofIκB-derived substrates, particularly IκB and NIK substrates. A widevariety of assays for binding agents are provided including labeled invitro protein-protein binding assays, immunoassays, cell based assays,etc. The methods are amenable to automated, cost-effective highthroughput screening of chemical libraries for lead compounds.Identified reagents find use in the pharmaceutical industries for animaland human trials; for example, the reagents may be derivatized andrescreened in in vitro and in vivo assays to optimize activity andminimize toxicity for pharmaceutical development.

In vitro binding assays employ a mixture of components including an IKKpolypeptide, which may be part of a fusion product with another peptideor polypeptide, e.g. a tag for detection or anchoring, etc. The assaymixtures comprise a natural intracellular IKK binding target. In aparticular embodiment, the binding target is a substrate comprising IκBserines 32 and/or 36. Such substrates comprise a IκBα, β or ε peptideincluding the serine 32 and/or 36 residue and at least 5, preferably atleast 10, and more preferably at least 20 naturally occurringimmediately flanking residues on each side (e.g. for serine 36 peptides,residues 26-46, 22-42, or 12-32 or 151-171 for IκBα, β, or ε-derivedsubstrates, respectively). While native full-length binding targets maybe used, it is frequently preferred to use portions (e.g. peptides)thereof so long as the portion provides binding affinity and avidity tothe subject IKK polypeptide conveniently measurable in the assay. Theassay mixture also comprises a candidate pharmacological agent.Candidate agents encompass numerous chemical classes, though typicallythey are organic compounds; preferably small organic compounds and areobtained from a wide variety of sources including libraries of syntheticor natural compounds. A variety of other reagents may also be includedin the mixture. These include reagents like ATP or ATP analogs (forkinase assays), salts, buffers, neutral proteins, e.g. albumin,detergents, protease inhibitors, nuclease inhibitors, antimicrobialagents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for thepresence of the candidate pharmacological agent, the IKK polypeptidespecifically binds the cellular binding target, portion or analog with areference binding affinity. The mixture components can be added in anyorder that provides for the requisite bindings and incubations may beperformed at any temperature which facilitates optimal binding.Incubation periods are likewise selected for optimal binding but alsominimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the IKK polypeptideand one or more binding targets is detected by any convenient way. ForIKK kinase assays, ‘binding’ is generally detected by a change in thephosphorylation of a IKK-α substrate. In this embodiment, kinaseactivity may quantified by the transfer to the substrate of a labeledphosphate, where the label may provide for direct detection asradioactivity, luminescence, optical or electron density, etc. orindirect detection such as an epitope tag, etc. A variety of methods maybe used to detect the label depending on the nature of the label andother assay components, e.g. through optical or electron density,radiative emissions, nonradiative energy transfers, etc. or indirectlydetected with antibody conjugates, etc.

A difference in the binding affinity of the IKK polypeptide to thetarget in the absence of the agent as compared with the binding affinityin the presence of the agent indicates that the agent modulates thebinding of the IKK polypeptide to the IKK binding target. Analogously,in the cell-based assay also described below, a difference inIKK-α-dependent transcriptional activation in the presence and absenceof an agent indicates the agent modulates IKK function. A difference, asused herein, is statistically significant and preferably represents atleast a 50%, more preferably at least a 90% difference.

The following experimental section and examples are offered by way ofillustration and not by way of limitation.

EXPERIMENTAL

Identification of IKK-α

To investigate the mechanism of NIK-mediated NF-κB activation, weidentified proteins that associate directly with NIK by yeast two-hybridprotein interaction cloning (Fields and Song, 1989). An expressionvector was generated that encodes NIK fused to the DNA-binding domain ofthe yeast transcription factor GAL4. This vector was used as bait in atwo-hybrid screen of a human B cell cDNA library. From approximately sixmillion transformants, eight positive clones were obtained, asdetermined by activation of his and lacZ reporter genes. Of theseclones, three encoded a member of the TRAF family, TRAF3 (Hu et al.,1994; Cheng et al., 1995; Mosialos et al., 1995; Sato et al., 1995) andone encoded a novel protein we call IKK-α. Retransformation into yeastcells verified the interaction between NIK and IKK-α. A full-lengthhuman IKK-α clone was isolated by screening a Jurkat cDNA library with aprobe generated from the 5′-end of the IKK-α two-hybrid clone. IKK-αcomprises an N-terminal serine-threonine kinase catalytic domain, aC-terminal helix-loop-helix domain and a leucine zipper-like amphipathicα-helix juxtaposed in between the helix-loop-helix and kinase domain.

Interaction of IKK-α and NIK in Human Cells

The interaction of IKK-α with NIK was confirmed in mammalian cellcoimmunoprecipitation assays. Human IKK-α containing an N-terminal Flagepitope tag was transiently coexpressed in 293 human embryonic kidneycells with Myc epitope-tagged NIK or HA epitope-tagged TRAF proteins.Cell lysates were immunoprecipitated using a monoclonal antibody againstthe Flag epitope, and coprecipitating NIK or TRAF proteins were detectedby immunoblot analysis with an anti-Myc or anti-HA monoclonalantibodies. In this assay, IKK-α was able to coprecipitate NIKconfirming the interaction between both proteins as detected for IKK-αby yeast two-hybrid analysis. Also, a deletion mutant IKK-α proteinlacking most of the N-terminal kinase domain (IKK-α₍₃₀₇₋₇₄₅₎) was ableto associate with NIK, indicating that the a-helical C-terminal half ofIKK-α mediates the interaction with NIK. In contrast to NIK, IKK-αfailed to associate with either TRAF2 or TRAF3. However, when NIK wascoexpressed with IKK-α and TRAF2, strong coprecipitation of TRAF2 withIKK-α was detected, indicating the formation of a ternary complexbetween IKK-α, NIK and TRAF2.

Effect of IKK-α and IKK-α Mutants on NF-κB Activation

To investigate a possible role for IKK-α in NF-κB activation, weexamined if transient overexpression of IKK-α might activate anNF-κB-dependent reporter gene. An E-selectin-luciferase reporterconstruct (Schindler and Baichwal, 1994) and a IKK-α expression vectorwere cotransfected into HeLa cells. IKK-α expression activated thereporter gene in a dose-dependent manner, with a maximal induction ofluciferase activity of about 6 to 7-fold compared to vector control.Similar results were obtained in 293 cells, where IKK-α overexpressioninduced reporter gene activity approximately 4-fold. TNF treatment didnot stimulate the weak NF-κB-inducing activity of overexpressed IKK-α inreporter gene assays. Thus, IKK-α induces NF-κB activation whenoverexpressed.

We next determined the effect of overexpression of kinase-inactiveIKK-α₍₃₀₇₋₇₄₅₎ that still associates with NIK on signal-induced NF-κBactivation in reporter gene assays in 293 cells. Overexpression ofIKK-α₍₃₀₇₋₇₄₅₎ blocked TNF- and IL-1-induced reporter gene activationsimilar to overexpression of NIK₍₆₂₄₋₉₄₇₎. IKK-α₍₃₀₇₋₇₄₅₎ was also foundto inhibited NF-κB-dependent reporter gene activity elicited byoverexpression of TRAF2, TRAF6 and NIK. Taken together these resultsdemonstrate that a catalytically inactive IKK-α mutant is adominant-negative inhibitor of TNF-, IL-1, TRAF- and NIK-induced NF-κBactivation. This indicates that IKK-α functions as a common mediator ofNF-κB activation by TNF and IL-1 downstream of NIK.

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EXAMPLES

1. Protocol for at IKK-α-IκBα phosphorylation assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

kinase: 10⁻⁸-10⁻⁵ M IKK-α (SEQ ID NO:4) at 20 μg/ml in PBS.

substrate: 10⁻⁷-10⁻⁴ M biotinylated substrate (21 residue peptideconsisting of residues 26-46 of human IκBα) at 40 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 20 mM HEPES pH 7.4,0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktailof protease inhibitors.

-[³²P]γ-ATP 10x stock: 2×10⁻⁵ M cold ATP with 100 μCi [³²P]γ-ATP. Placein the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2 mM NaVo₃ (Sigma #S-6508) in 10 ml of PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 40 μl biotinylated substrate (2-200 pmoles/40 ul in assay buffer)

Add 40 μl kinase (0.1-10 pmoles/40 ul in assay buffer)

Add 10 μl compound or extract.

Add 10 μl [³²P]γ-ATP 10x stock.

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. cold ATP at 80% inhibition.

2. Protocol for high throughput IKK-α-NIK binding assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol,0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of proteaseinhibitors.

³³P IKK-α polypeptide 10x stock: 10⁻⁸-10⁻⁶ M “cold” IKK-α supplementedwith 200,000-250,000 cpm of labeled IKK-α (Beckman counter). Place inthe 4° C. microfridge during screening.

Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2 mM NaVO₃ (Sigma #S-6508) in 10 ml of PBS.

NIK: 10⁻⁷-10⁻⁵ M biotinylated NIK in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³P-IKK-α (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹-10⁻⁷ M finalconc).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μM biotinylated NIK (0.1-10 pmoles/40 ul in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μM PBS.

Add 150 μM scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. Soluble (non-biotinylated NIK) at 80% inhibition.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

5 2268 base pairs nucleic acid double linear cDNA unknown 1 ATGAGCTGGTCACCTTCCCT GACAACGCAG ACATGTGGGG CCTGGGAAAT GAAAGAGCGC 60 CTTGGGACAGGGGGATTTGG AAATGTCATC CGATGGCACA ATCAGGAAAC AGGTGAGCAG 120 ATTGCCATCAAGCAGTGCCG GCAGGAGCTC AGCCCCCGGA ACCGAGAGCG GTGGTGCCTG 180 GAGATCCAGATCATGAGAAG GCTGACCCAC CCCAATGTGG TGGCTGCCCG AGATGTCCCT 240 GAGGGGATGCAGAACTTGGC GCCCAATGAC CTGCCCCTGC TGGCCATGGA GTACTGCCAA 300 GGAGGAGATCTCCGGAAGTA CCTGAACCAG TTTGAGAACT GCTGTGGTCT GCGGGAAGGT 360 GCCATCCTCACCTTGCTGAG TGACATTGCC TCTGCGCTTA GATACCTTCA TGAAAACAGA 420 ATCATCCATCGGGATCTAAA GCCAGAAAAC ATCGTCCTGC AGCAAGGAGA ACAGAGGTTA 480 ATACACAAAATTATTGACCT AGGATATGCC AAGGAGCTGG ATCAGGGCAG TCTTTGCACA 540 TCATTCGTGGGGACCCTGCA GTACCTGGCC CCAGAGCTAC TGGAGCAGCA GAAGTACACA 600 GTGACCGTCGACTACTGGAG CTTCGGCACC CTGGCCTTTG AGTGCATCAC GGGCTTCCGG 660 CCCTTCCTCCCCAACTGGCA GCCCGTGCAG TGGCATTCAA AAGTGCGGCA GAAGAGTGAG 720 GTGGACATTGTTGTTAGCGA AGACTTGAAT GGAACGGTGA AGTTTTCAAG CTCTTTACCC 780 TACCCCAATAATCTTAACAG TGTCCTGGCT GAGCGACTGG AGAAGTGGCT GCAACTGATG 840 CTGATGTGGCACCCCCGACA GAGGGGCACG GATCCCACGT ATGGGCCCAA TGGCTGCTTC 900 AAGGCCCTGGATGACATCTT AAACTTAAAG CTGGTTCATA TCTTGAACAT GGTCACGGGC 960 ACCATCCACACCTACCCTGT GACAGAGGAT GAGAGTCTGC AGAGCTTGAA GGCCAGAATC 1020 CAACAGGACACGGGCATCCC AGAGGAGGAC CAGGAGCTGC TGCAGGAAGC GGGCCTGGCG 1080 TTGATCCCCGATAAGCCTGC CACTCAGTGT ATTTCAGACG GCAAGTTAAA TGAGGGCCAC 1140 ACATTGGACATGGATCTTGT TTTTCTCTTT GACAACAGTA AAATCACCTA TGAGACTCAG 1200 ATCTCCCCACGGCCCCAACC TGAAAGTGTC AGCTGTATCC TTCAAGAGCC CAAGAGGAAT 1260 CTCGCCTTCTTCCAGCTGAG GAAGGTGTGG GGCCAGGTCT GGCACAGCAT CCAGACCCTG 1320 AAGGAAGATTGCAACCGGCT GCAGCAGGGA CAGCGAGCCG CCATGATGAA TCTCCTCCGA 1380 AACAACAGCTGCCTCTCCAA AATGAAGAAT TCCATGGCTT CCATGTCTCA GCAGCTCAAG 1440 GCCAAGTTGGATTTCTTCAA AACCAGCATC CAGATTGACC TGGAGAAGTA CAGCGAGCAA 1500 ACCGAGTTTGGGATCACATC AGATAAACTG CTGCTGGCCT GGAGGGAAAT GGAGCAGGCT 1560 GTGGAGCTCTGTGGGCGGGA GAACGAAGTG AAACTCCTGG TAGAACGGAT GATGGCTCTG 1620 CAGACCGACATTGTGGACTT ACAGAGGAGC CCCATGGGCC GGAAGCAGGG GGGAACGCTG 1680 GACGACCTAGAGGAGCAAGC AAGGGAGCTG TACAGGAGAC TAAGGGAAAA ACCTCGAGAC 1740 CAGCGAACTGAGGGTGACAG TCAGGAAATG GTACGGCTGC TGCTTCAGGC AATTCAGAGC 1800 TTCGAGAAGAAAGTGCGAGT GATCTATACG CAGCTCAGTA AAACTGTGGT TTGCAAGCAG 1860 AAGGCGCTGGAACTGTTGCC CAAGGTGGAA GAGGTGGTGA GCTTAATGAA TGAGGATGAG 1920 AAGACTGTTGTCCGGCTGCA GGAGAAGCGG CAGAAGGAGC TCTGGAATCT CCTGAAGATT 1980 GCTTGTAGCAAGGTCCGTGG TCCTGTCAGT GGAAGCCCGG ATAGCATGAA TGCCTCTCGA 2040 CTTAGCCAGCCTGGGCAGCT GATGTCTCAG CCCTCCACGG CCTCCAACAG CTTACCTGAG 2100 CCAGCCAAGAAGAGTGAAGA ACTGGTGGCT GAAGCACATA ACCTCTGCAC CCTGCTAGAA 2160 AATGCCATACAGGACACTGT GAGGGAACAA GACCAGAGTT TCACGGCCCT AGACTGGAGC 2220 TGGTTACAGACGGAAGAAGA AGAGCACAGC TGCCTGGAGC AGGCCTCA 2268 756 amino acids aminoacid single linear peptide unknown 2 Met Ser Trp Ser Pro Ser Leu Thr ThrGln Thr Cys Gly Ala Trp Glu 1 5 10 15 Met Lys Glu Arg Leu Gly Thr GlyGly Phe Gly Asn Val Ile Arg Trp 20 25 30 His Asn Gln Glu Thr Gly Glu GlnIle Ala Ile Lys Gln Cys Arg Gln 35 40 45 Glu Leu Ser Pro Arg Asn Arg GluArg Trp Cys Leu Glu Ile Gln Ile 50 55 60 Met Arg Arg Leu Thr His Pro AsnVal Val Ala Ala Arg Asp Val Pro 65 70 75 80 Glu Gly Met Gln Asn Leu AlaPro Asn Asp Leu Pro Leu Leu Ala Met 85 90 95 Glu Tyr Cys Gln Gly Gly AspLeu Arg Lys Tyr Leu Asn Gln Phe Glu 100 105 110 Asn Cys Cys Gly Leu ArgGlu Gly Ala Ile Leu Thr Leu Leu Ser Asp 115 120 125 Ile Ala Ser Ala LeuArg Tyr Leu His Glu Asn Arg Ile Ile His Arg 130 135 140 Asp Leu Lys ProGlu Asn Ile Val Leu Gln Gln Gly Glu Gln Arg Leu 145 150 155 160 Ile HisLys Ile Ile Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly 165 170 175 SerLeu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu 180 185 190Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe 195 200205 Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Phe Arg Pro Phe Leu Pro 210215 220 Asn Trp Gln Pro Val Gln Trp His Ser Lys Val Arg Gln Lys Ser Glu225 230 235 240 Val Asp Ile Val Val Ser Glu Asp Leu Asn Gly Thr Val LysPhe Ser 245 250 255 Ser Ser Leu Pro Tyr Pro Asn Asn Leu Asn Ser Val LeuAla Glu Arg 260 265 270 Leu Glu Lys Trp Leu Gln Leu Met Leu Met Trp HisPro Arg Gln Arg 275 280 285 Gly Thr Asp Pro Thr Tyr Gly Pro Asn Gly CysPhe Lys Ala Leu Asp 290 295 300 Asp Ile Leu Asn Leu Lys Leu Val His IleLeu Asn Met Val Thr Gly 305 310 315 320 Thr Ile His Thr Tyr Pro Val ThrGlu Asp Glu Ser Leu Gln Ser Leu 325 330 335 Lys Ala Arg Ile Gln Gln AspThr Gly Ile Pro Glu Glu Asp Gln Glu 340 345 350 Leu Leu Gln Glu Ala GlyLeu Ala Leu Ile Pro Asp Lys Pro Ala Thr 355 360 365 Gln Cys Ile Ser AspGly Lys Leu Asn Glu Gly His Thr Leu Asp Met 370 375 380 Asp Leu Val PheLeu Phe Asp Asn Ser Lys Ile Thr Tyr Glu Thr Gln 385 390 395 400 Ile SerPro Arg Pro Gln Pro Glu Ser Val Ser Cys Ile Leu Gln Glu 405 410 415 ProLys Arg Asn Leu Ala Phe Phe Gln Leu Arg Lys Val Trp Gly Gln 420 425 430Val Trp His Ser Ile Gln Thr Leu Lys Glu Asp Cys Asn Arg Leu Gln 435 440445 Gln Gly Gln Arg Ala Ala Met Met Asn Leu Leu Arg Asn Asn Ser Cys 450455 460 Leu Ser Lys Met Lys Asn Ser Met Ala Ser Met Ser Gln Gln Leu Lys465 470 475 480 Ala Lys Leu Asp Phe Phe Lys Thr Ser Ile Gln Ile Asp LeuGlu Lys 485 490 495 Tyr Ser Glu Gln Thr Glu Phe Gly Ile Thr Ser Asp LysLeu Leu Leu 500 505 510 Ala Trp Arg Glu Met Glu Gln Ala Val Glu Leu CysGly Arg Glu Asn 515 520 525 Glu Val Lys Leu Leu Val Glu Arg Met Met AlaLeu Gln Thr Asp Ile 530 535 540 Val Asp Leu Gln Arg Ser Pro Met Gly ArgLys Gln Gly Gly Thr Leu 545 550 555 560 Asp Asp Leu Glu Glu Gln Ala ArgGlu Leu Tyr Arg Arg Leu Arg Glu 565 570 575 Lys Pro Arg Asp Gln Arg ThrGlu Gly Asp Ser Gln Glu Met Val Arg 580 585 590 Leu Leu Leu Gln Ala IleGln Ser Phe Glu Lys Lys Val Arg Val Ile 595 600 605 Tyr Thr Gln Leu SerLys Thr Val Val Cys Lys Gln Lys Ala Leu Glu 610 615 620 Leu Leu Pro LysVal Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu 625 630 635 640 Lys ThrVal Val Arg Leu Gln Glu Lys Arg Gln Lys Glu Leu Trp Asn 645 650 655 LeuLeu Lys Ile Ala Cys Ser Lys Val Arg Gly Pro Val Ser Gly Ser 660 665 670Pro Asp Ser Met Asn Ala Ser Arg Leu Ser Gln Pro Gly Gln Leu Met 675 680685 Ser Gln Pro Ser Thr Ala Ser Asn Ser Leu Pro Glu Pro Ala Lys Lys 690695 700 Ser Glu Glu Leu Val Ala Glu Ala His Asn Leu Cys Thr Leu Leu Glu705 710 715 720 Asn Ala Ile Gln Asp Thr Val Arg Glu Gln Asp Gln Ser PheThr Ala 725 730 735 Leu Asp Trp Ser Trp Leu Gln Thr Glu Glu Glu Glu HisSer Cys Leu 740 745 750 Glu Gln Ala Ser 755 2238 base pairs nucleic aciddouble linear cDNA unknown 3 ATGGAGCGGC CCCCGGGGCT GCGGCCGGGC GCGGGCGGGCCCTGGGAGAT GCGGGAGCGG 60 CTGGGCACCG GCGGCTTCGG GAACGTCTGT CTGTACCAGCATCGGGAACT TGATCTCAAA 120 ATAGCAATTA AGTCTTGTCG CCTAGAGCTA AGTACCAAAAACAGAGAACG ATGGTGCCAT 180 GAAATCCAGA TTATGAAGAA GTTGAACCAT GCCAATGTTGTAAAGGCCTG TGATGTTCCT 240 GAAGAATTGA ATATTTTGAT TCATGATGTG CCTCTTCTAGCAATGGAATA CTGTTCTGGA 300 GGAGATCTCC GAAAGCTGCT CAACAAACCA GAAAATTGTTGTGGACTTAA AGAAAGCCAG 360 ATACTTTCTT TACTAAGTGA TATAGGGTCT GGGATTCGATATTTGCATGA AAACAAAATT 420 ATACATCGAG ATCTAAAACC TGAAAACATA GTTCTTCAGGATGTTGGTGG AAAGATAATA 480 CATAAAATAA TTGATCTGGG ATATGCCAAA GATGTTGATCAAGGAAGTCT GTGTACATCT 540 TTTGTGGGAA CACTGCAGTA TCTGGCCCCA GAGCTCTTTGAGAATAAGCC TTACACAGCC 600 ACTGTTGATT ATTGGAGCTT TGGGACCATG GTATTTGAATGTATTGCTGG ATATAGGCCT 660 TTTTTGCATC ATCTGCAGCC ATTTACCTGG CATGAGAAGATTAAGAAGAA GGATCCAAAG 720 TGTATATTTG CATGTGAAGA GATGTCAGGA GAAGTTCGGTTTAGTAGCCA TTTACCTCAA 780 CCAAATAGCC TTTGTAGTTT AATAGTAGAA CCCATGGAAAACTGGCTACA GTTGATGTTG 840 AATTGGGACC CTCAGCAGAG AGGAGGACCT GTTGACCTTACTTTGAAGCA GCCAAGATGT 900 TTTGTATTAA TGGATCACAT TTTGAATTTG AAGATAGTACACATCCTAAA TATGACTTCT 960 GCAAAGATAA TTTCTTTTCT GTTACCACCT GATGAAAGTCTTCATTCACT ACAGTCTCGT 1020 ATTGAGCGTG AAACTGGAAT AAATACTGGT TCTCAAGAACTTCTTTCAGA GACAGGAATT 1080 TCTCTGGATC CTCGGAAACC AGCCTCTCAA TGTGTTCTAGATGGAGTTAG AGGCTGTGAT 1140 AGCTATATGG TTTATTTGTT TGATAAAAGT AAAACTGTATATGAAGGGCC ATTTGCTTCC 1200 AGAAGTTTAT CTGATTGTGT AAATTATATT GTACAGGACAGCAAAATACA GCTTCCAATT 1260 ATACAGCTGC GTAAAGTGTG GGCTGAAGCA GTGCACTATGTGTCTGGACT AAAAGAAGAC 1320 TATAGCAGGC TCTTTCAGGG ACAAAGGGCA GCAATGTTAAGTCTTCTTAG ATATAATGCT 1380 AACTTAACAA AAATGAAGAA CACTTTGATC TCAGCATCACAACAACTGAA AGCTAAATTG 1440 GAGTTTTTTC ACAAAAGCAT TCAGCTTGAC TTGGAGAGATACAGCGAGCA GATGACGTAT 1500 GGGATATCTT CAGAAAAAAT GCTAAAAGCA TGGAAAGAAATGGAAGAAAA GGCCATCCAC 1560 TATGCTGAGG TTGGTGTCAT TGGATACCTG GAGGATCAGATTATGTCTTT GCATGCTGAA 1620 ATCATGGAGC TACAGAAGAG CCCCTATGGA AGACGTCAGGGAGACTTGAT GGAATCTCTG 1680 GAACAGCGTG CCATTGATCT ATATAAGCAG TTAAAACACAGACCTTCAGA TCACTCCTAC 1740 AGTGACAGCA CAGAGATGGT GAAAATCATT GTGCACACTGTGCAGAGTCA GGACCGTGTG 1800 CTCAAGGAGC TGTTTGGTCA TTTGAGCAAG TTGTTGGGCTGTAAGCAGAA GATTATTGAT 1860 CTACTCCCTA AGGTGGAAGT GGCCCTCAGT AATATCAAAGAAGCTGACAA TACTGTCATG 1920 TTCATGCAGG GAAAAAGGCA GAAAGAAATA TGGCATCTCCTTAAAATTGC CTGTACACAG 1980 AGTTCTGCCC GGTCCCTTGT AGGATCCAGT CTAGAAGGTGCAGTAACCCC TCAGACATCA 2040 GCATGGCTGC CCCCGACTTC AGCAGAACAT GATCATTCTCTGTCATGTGT GGTAACTCCT 2100 CAAGATGGGG AGACTTCAGC ACAAATGATA GAAGAAAATTTGAACTGCCT TGGCCATTTA 2160 AGCACTATTA TTCATGAGGC AAATGAGGAA CAGGGCAATAGTATGATGAA TCTTGATTGG 2220 AGTTGGTTAA CAGAATGA 2238 745 amino acidsamino acid single linear peptide unknown 4 Met Glu Arg Pro Pro Gly LeuArg Pro Gly Ala Gly Gly Pro Trp Glu 1 5 10 15 Met Arg Glu Arg Leu GlyThr Gly Gly Phe Gly Asn Val Cys Leu Tyr 20 25 30 Gln His Arg Glu Leu AspLeu Lys Ile Ala Ile Lys Ser Cys Arg Leu 35 40 45 Glu Leu Ser Thr Lys AsnArg Glu Arg Trp Cys His Glu Ile Gln Ile 50 55 60 Met Lys Lys Leu Asn HisAla Asn Val Val Lys Ala Cys Asp Val Pro 65 70 75 80 Glu Glu Leu Asn IleLeu Ile His Asp Val Pro Leu Leu Ala Met Glu 85 90 95 Tyr Cys Ser Gly GlyAsp Leu Arg Lys Leu Leu Asn Lys Pro Glu Asn 100 105 110 Cys Cys Gly LeuLys Glu Ser Gln Ile Leu Ser Leu Leu Ser Asp Ile 115 120 125 Gly Ser GlyIle Arg Tyr Leu His Glu Asn Lys Ile Ile His Arg Asp 130 135 140 Leu LysPro Glu Asn Ile Val Leu Gln Asp Val Gly Gly Lys Ile Ile 145 150 155 160His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Asp Val Asp Gln Gly Ser 165 170175 Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu 180185 190 Phe Glu Asn Lys Pro Tyr Thr Ala Thr Val Asp Tyr Trp Ser Phe Gly195 200 205 Thr Met Val Phe Glu Cys Ile Ala Gly Tyr Arg Pro Phe Leu HisHis 210 215 220 Leu Gln Pro Phe Thr Trp His Glu Lys Ile Lys Lys Lys AspPro Lys 225 230 235 240 Cys Ile Phe Ala Cys Glu Glu Met Ser Gly Glu ValArg Phe Ser Ser 245 250 255 His Leu Pro Gln Pro Asn Ser Leu Cys Ser LeuIle Val Glu Pro Met 260 265 270 Glu Asn Trp Leu Gln Leu Met Leu Asn TrpAsp Pro Gln Gln Arg Gly 275 280 285 Gly Pro Val Asp Leu Thr Leu Lys GlnPro Arg Cys Phe Val Leu Met 290 295 300 Asp His Ile Leu Asn Leu Lys IleVal His Ile Leu Asn Met Thr Ser 305 310 315 320 Ala Lys Ile Ile Ser PheLeu Leu Pro Pro Asp Glu Ser Leu His Ser 325 330 335 Leu Gln Ser Arg IleGlu Arg Glu Thr Gly Ile Asn Thr Gly Ser Gln 340 345 350 Glu Leu Leu SerGlu Thr Gly Ile Ser Leu Asp Pro Arg Lys Pro Ala 355 360 365 Ser Gln CysVal Leu Asp Gly Val Arg Gly Cys Asp Ser Tyr Met Val 370 375 380 Tyr LeuPhe Asp Lys Ser Lys Thr Val Tyr Glu Gly Pro Phe Ala Ser 385 390 395 400Arg Ser Leu Ser Asp Cys Val Asn Tyr Ile Val Gln Asp Ser Lys Ile 405 410415 Gln Leu Pro Ile Ile Gln Leu Arg Lys Val Trp Ala Glu Ala Val His 420425 430 Tyr Val Ser Gly Leu Lys Glu Asp Tyr Ser Arg Leu Phe Gln Gly Gln435 440 445 Arg Ala Ala Met Leu Ser Leu Leu Arg Tyr Asn Ala Asn Leu ThrLys 450 455 460 Met Lys Asn Thr Leu Ile Ser Ala Ser Gln Gln Leu Lys AlaLys Leu 465 470 475 480 Glu Phe Phe His Lys Ser Ile Gln Leu Asp Leu GluArg Tyr Ser Glu 485 490 495 Gln Met Thr Tyr Gly Ile Ser Ser Glu Lys MetLeu Lys Ala Trp Lys 500 505 510 Glu Met Glu Glu Lys Ala Ile His Tyr AlaGlu Val Gly Val Ile Gly 515 520 525 Tyr Leu Glu Asp Gln Ile Met Ser LeuHis Ala Glu Ile Met Glu Leu 530 535 540 Gln Lys Ser Pro Tyr Gly Arg ArgGln Gly Asp Leu Met Glu Ser Leu 545 550 555 560 Glu Gln Arg Ala Ile AspLeu Tyr Lys Gln Leu Lys His Arg Pro Ser 565 570 575 Asp His Ser Tyr SerAsp Ser Thr Glu Met Val Lys Ile Ile Val His 580 585 590 Thr Val Gln SerGln Asp Arg Val Leu Lys Glu Leu Phe Gly His Leu 595 600 605 Ser Lys LeuLeu Gly Cys Lys Gln Lys Ile Ile Asp Leu Leu Pro Lys 610 615 620 Val GluVal Ala Leu Ser Asn Ile Lys Glu Ala Asp Asn Thr Val Met 625 630 635 640Phe Met Gln Gly Lys Arg Gln Lys Glu Ile Trp His Leu Leu Lys Ile 645 650655 Ala Cys Thr Gln Ser Ser Ala Arg Ser Leu Val Gly Ser Ser Leu Glu 660665 670 Gly Ala Val Thr Pro Gln Thr Ser Ala Trp Leu Pro Pro Thr Ser Ala675 680 685 Glu His Asp His Ser Leu Ser Cys Val Val Thr Pro Gln Asp GlyGlu 690 695 700 Thr Ser Ala Gln Met Ile Glu Glu Asn Leu Asn Cys Leu GlyHis Leu 705 710 715 720 Ser Thr Ile Ile His Glu Ala Asn Glu Glu Gln GlyAsn Ser Met Met 725 730 735 Asn Leu Asp Trp Ser Trp Leu Thr Glu 740 7452146 base pairs nucleic acid double linear cDNA unknown 5 GTACCAGCATCGGGAACTTG ATCTCAAAAT AGCAATTAAG TCTTGTCGCC TAGAGCTAAG 60 TACCAAAAACAGAGAACGAT GGTGCCATGA AATCCAGATT ATGAAGAAGT TGAACCATGC 120 CAATGTTGTAAAGGCCTGTG ATGTTCCTGA AGAATTGAAT ATTTTGATTC ATGATGTGCC 180 TCTTCTAGCAATGGAATACT GTTCTGGAGG AGATCTCCGA AAGCTGCTCA ACAAACCAGA 240 AAATTGTTGTGGACTTAAAG AAAGCCAGAT ACTTTCTTTA CTAAGTGATA TAGGGTCTGG 300 GATTCGATATTTGCATGAAA ACAAAATTAT ACATCGAGAT CTAAAACCTG AAAACATAGT 360 TCTTCAGGATGTTGGTGGAA AGATAATACA TAAAATAATT GATCTGGGAT ATGCCAAAGA 420 TGTTGATCAAGGAAGTCTGT GTACATCTTT TGTGGGAACA CTGCAGTATC TGGCCCCAGA 480 GCTCTTTGAGAATAAGCCTT ACACAGCCAC TGTTGATTAT TGGAGCTTTG GGACCATGGT 540 ATTTGAATGTATTGCTGGAT ATAGGCCTTT TTTGCATCAT CTGCAGCCAT TTACCTGGCA 600 TGAGAAGATTAAGAAGAAGG ATCCAAAGTG TATATTTGCA TGTGAAGAGA TGTCAGGAGA 660 AGTTCGGTTTAGTAGCCATT TACCTCAACC AAATAGCCTT TGTAGTTTAA TAGTAGAACC 720 CATGGAAAACTGGCTACAGT TGATGTTGAA TTGGGACCCT CAGCAGAGAG GAGGACCTGT 780 TGACCTTACTTTGAAGCAGC CAAGATGTTT TGTATTAATG GATCACATTT TGAATTTGAA 840 GATAGTACACATCCTAAATA TGACTTCTGC AAAGATAATT TCTTTTCTGT TACCACCTGA 900 TGAAAGTCTTCATTCACTAC AGTCTCGTAT TGAGCGTGAA ACTGGAATAA ATACTGGTTC 960 TCAAGAACTTCTTTCAGAGA CAGGAATTTC TCTGGATCCT CGGAAACCAG CCTCTCAATG 1020 TGTTCTAGATGGAGTTAGAG GCTGTGATAG CTATATGGTT TATTTGTTTG ATAAAAGTAA 1080 AACTGTATATGAAGGGCCAT TTGCTTCCAG AAGTTTATCT GATTGTGTAA ATTATATTGT 1140 ACAGGACAGCAAAATACAGC TTCCAATTAT ACAGCTGCGT AAAGTGTGGG CTGAAGCAGT 1200 GCACTATGTGTCTGGACTAA AAGAAGACTA TAGCAGGCTC TTTCAGGGAC AAAGGGCAGC 1260 AATGTTAAGTCTTCTTAGAT ATAATGCTAA CTTAACAAAA ATGAAGAACA CTTTGATCTC 1320 AGCATCACAACAACTGAAAG CTAAATTGGA GTTTTTTCAC AAAAGCATTC AGCTTGACTT 1380 GGAGAGATACAGCGAGCAGA TGACGTATGG GATATCTTCA GAAAAAATGC TAAAAGCATG 1440 GAAAGAAATGGAAGAAAAGG CCATCCACTA TGCTGAGGTT GGTGTCATTG GATACCTGGA 1500 GGATCAGATTATGTCTTTGC ATGCTGAAAT CATGGAGCTA CAGAAGAGCC CCTATGGAAG 1560 ACGTCAGGGAGACTTGATGG AATCTCTGGA ACAGCGTGCC ATTGATCTAT ATAAGCAGTT 1620 AAAACACAGACCTTCAGATC ACTCCTACAG TGACAGCACA GAGATGGTGA AAATCATTGT 1680 GCACACTGTGCAGAGTCAGG ACCGTGTGCT CAAGGAGCGT TTTGGTCATT TGAGCAAGTT 1740 GTTGGGCTGTAAGCAGAAGA TTATTGATCT ACTCCCTAAG GTGGAAGTGG CCCTCAGTAA 1800 TATCAAAGAAGCTGACAATA CTGTCATGTT CATGCAGGGA AAAAGGCAGA AAGAAATATG 1860 GCATCTCCTTAAAATTGCCT GTACACAGAG TTCTGCCCGC TCTCTTGTAG GATCCAGTCT 1920 AGAAGGTGCAGTAACCCCTC AAGCATACGC ATGGCTGGCC CCCGACTTAG CAGAACATGA 1980 TCATTCTCTGTCATGTGTGG TAACTCCTCA AGATGGGGAG ACTTCAGCAC AAATGATAGA 2040 AGAAAATTTGAACTGCCTTG GCCATTTAAG CACTATTATT CATGAGGCAA ATGAGGAACA 2100 GGGCAATAGTATGATGAATC TTGATTGGAG TTGGTTAACA GAATGA 2146

What is claimed is:
 1. A method of screening for an agent whichmodulates the interaction of an IKK polypeptide to a binding target,said method comprising the steps of: incubating a mixture comprising: anisolated polypeptide comprising at least 10 consecutive residues of theamino acid sequence set forth as SEQ ID NO:4, which consecutive aminoacid residues comprise at least one of the amino acid residues 679, 680,684, 686 and 687 of SEQ ID NO:4, a binding target of said polypeptide,and a candidate agent; under conditions whereby, but for the presence ofsaid agent, said polypeptide specifically binds said binding target at areference affinity; detecting the binding affinity of said polypeptideto said binding target to determine an agent-biased affinity, wherein adifference between the agent-biased affinity and the reference affinityindicates that said agent modulates the binding of said polypeptide tosaid binding target.
 2. A method according to claim 1, wherein saidpolypeptide has an activity selected from at least one of: a kinase orkinase inhibitory activity, a NIK-binding or binding inhibitoryactivity, an IκB-binding or binding inhibitory activity and an NFκBactivating or inhibitory activity.
 3. A method according to claim 1,wherein the consecutive amino acid residues comprise the amino acidresidue 679 of SEQ ID NO:4.
 4. A method according to claim 1, whereinthe consecutive amino acid residues comprise the amino acid residue 680of SEQ ID NO:4.
 5. A method according to claim 1, wherein theconsecutive amino acid residues comprise the amino acid residue 684 ofSEQ ID NO:4.
 6. A method according to claim 1, wherein the consecutiveamino acid residues comprise the amino acid residue 686 of SEQ ID NO:4.7. A method according to claim 1, wherein the consecutive amino acidresidues comprise the amino acid residue 687 of SEQ ID NO:4.
 8. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues 601-681 of SEQ ID NO:4.
 9. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues 604-679 of SEQ ID NO:4.
 10. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues 670-687 of SEQ ID NO:4.
 11. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues 679-687 of SEQ ID NO:4.
 12. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues 680-690 of SEQ ID NO:4.
 13. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues 684-695 of SEQ ID NO:4.
 14. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues 686-699 of SEQ ID NO:4.
 15. A methodaccording to claim 1, wherein the consecutive amino acid residuescomprise the amino acid residues of SEQ ID NO:4.
 16. A method accordingto claim 1, wherein said binding target is a natural intracellularsubstrate and said reference and agent-biased binding affinity isdetected as phosphorylation of said substrate.
 17. A method of screeningfor an agent which modulates the interaction of an IKK polypeptide to abinding target, said method comprising the steps of: incubating amixture comprising: an isolated polypeptide comprising at least 10consecutive residues of the amino acid sequence set forth as SEQ IDNO:4, which consecutive amino acid residues (a) comprise at least one ofthe amino acid residues 679, 680, 684, 686 and 687 of SEQ ID NO:4 and(b) retain IκB kinase activity, an IκB polypeptide comprising at least asix residue domain of a natural IκB comprising at least one of Ser32 andSer36, and a candidate agent; under conditions whereby, but for thepresence of said agent, said polypeptide specifically phosphorylatessaid IκB polypeptide at at least one of said Ser32 and Ser36 at areference activity; detecting the polypeptide-induced phosphorylation ofsaid IκB polypeptide at at least one of said Ser32 and Ser36 todetermine an agent-biased activity, wherein a difference between theagent-biased activity and the reference activity indicates that saidagent modulates the ability of said polypeptide to specificallyphosphorylate a IκB polypeptide.
 18. A method according to claim 17,wherein the consecutive amino acid residues comprise the amino acidresidue 679 of SEQ ID NO:4.
 19. A method according to claim 17, whereinthe consecutive amino acid residues comprise the amino acid residue 680of SEQ ID NO:4.
 20. A method according to claim 17, wherein theconsecutive amino acid residues comprise the amino acid residue 684 ofSEQ ID NO:4.
 21. A method according to claim 17, wherein the consecutiveamino acid residues comprise the amino acid residue 686 of SEQ ID NO:4.22. A method according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residue 687 of SEQ ID NO:4.
 23. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues 601-681 of SEQ ID NO:4.
 24. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues 604-679 of SEQ ID NO:4.
 25. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues 670-687 of SEQ ID NO:4.
 26. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues 679-687 of SEQ ID NO:4.
 27. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues 680-690 of SEQ ID NO:4.
 28. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues 684-695 of SEQ ID NO:4.
 29. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues 686-699 of SEQ ID NO:4.
 30. Amethod according to claim 17, wherein the consecutive amino acidresidues comprise the amino acid residues of SEQ ID NO:4.
 31. A methodaccording to claim 1, wherein said polypeptide has a kinase activity.32. A method according to claim 1, wherein said polypeptide has a kinaseinhibitory activity.
 33. A method according to claim 1, wherein saidpolypeptide has a NIK-binding activity.
 34. A method according to claim1, wherein said polypeptide has a NIK-binding inhibitory activity.
 35. Amethod according to claim 1, wherein said polypeptide has an IκB-bindingactivity.
 36. A method according to claim 1, wherein said polypeptidehas an IκB-binding inhibitory activity.
 37. A method according to claim1, wherein said polypeptide has an NFκB activating activity.
 38. Amethod according to claim 1, wherein said polypeptide has an NFκBinhibitory activity.
 39. A method according to claim 15, wherein saidpolypeptide has an activity selected from at least one of: a NIK-bindingor binding inhibitory activity, an IκB-binding or binding inhibitoryactivity and an NFκB activating or inhibitory activity.
 40. A methodaccording to claim 15, wherein said polypeptide has a NIK-bindingactivity.
 41. A method according to claim 15, wherein said polypeptidehas a NIK-binding inhibitory activity.
 42. A method according to claim15, wherein said polypeptide has an IκB-binding activity.
 43. A methodaccording to claim 15, wherein said polypeptide has an IκB-bindinginhibitory activity.
 44. A method according to claim 15, wherein saidpolypeptide has an NFκB activating activity.
 45. A method according toclaim 15, wherein said polypeptide has an NFκB inhibitory activity. 46.A method according to claim 1, wherein the polypeptide is purified. 47.A method according to claim 2, wherein the polypeptide is purified. 48.A method according to claim 17, wherein the polypeptide is purified. 49.A method according to claim 39, wherein the polypeptide is purified.